The vertebrate antibody repertoire was formed by the duplication and diversification of ancestral genes of a heterodimer of two immunoglobulin (Ig) folds. The diversity generated by the immune system relies not only on the germline gene families of Ig genes, but from the recombination of subdomain exons in vivo during B- and T-cell development to form numerous unique lineages with additional diversity at the exon boundaries that occur at surface-exposed loops of the Ig protein. This process of recombination is called V(D)J recombination, so called after the two variable light (VL) and three variable heavy (VH) exons that recombine to form the N-terminal antigen binding domains of the light chain and heavy chain of the antibody, respectively. However, as the duplicated genes diverged from their ancestral pair, the cumulated effect of mutations has resulted in a less-than-perfect interfacial fit between heterodimer units of the variable domains. Selection pressure is not applied to any one gene, but to the family as a whole. Thus, maximum diversity, which is a good thing for the immune system, can result in less-than-ideal folding stabilities for individual family members. Furthermore, the binding domains themselves may have different folding stabilities. The requirement to form a functional heterodimer from numerous diverged subunits is compensated for by the presence of conserved disulphide bonds between the Beta-sheets of the domains. However, the interface may still not be a stable fit, requiring a folding checkpoint in the ER.
As a result of the ‘consensus’ approach to a protein fit applied by the antibody variable domains, some pairings have a low folding stability and propensity for either poor expression in bacterial/mammalian hosts, and a propensity to aggregate. Furthermore, in almost all cases, there is a total requirement for the inter-sheet disulphide bonds to be formed within the VL and VH domains. This necessitates that for expression of antibody libraries in a bacterial host such as E. coli the antibody is expressed in the periplasm of the cell, an oxidizing space that has disulphide chaperones, and often as a fusion between the VL and VH domains (single chain antibody; scFv). However, export to the periplasm requires the excretion through the inner membrane, which is saturated at the levels desired for high expression of the antibody, resulting in far lower yields than cytoplasmic expression.
In addition to the advantage of cheaper production of scFv antibodies in the E. coli cytoplasm, an antibody scaffold that is competent to fold in a reducing environment would also be able to be used as an affinity reagent in the mammalian cytoplasm. This would enable the extension of the uses of antibodies as scientific reagents in the cytoplasm or nucleus for imaging or blocking protein function, and similarly in therapeutics and diagnostics.
As almost all mammalian antibodies are insoluble in the cytoplasm, groups have searched for the rare combinations of genes that fold to form a stable heterodimer to use as a scaffold for building further diversity. The approach taken to find cytoplasmically soluble antibodies is either the happenstance observation that an antibody clone is stably expressed in the cytoplasm (Tavladoraki et al., 1999; Vaccaro et al., 2006) which may form the basis for an intracellular antibody (“intrabody”) scaffold, or, alternatively, an evolutionary approach may be taken to evolve a scFv gene towards stability, either in vivo (Martineau et al., 1998; Visintin et al., 1999; Auf der Maur et al., 2002; Fisher and DeLisa, 2009) or in vitro (Contreras-Martinez and Delisa, 2007; Jermutus L., et al. 2001). Furthermore, single domain antibodies, where only a single, unpaired, variable domain binds to the target antigen, have proven to be soluble and stable in the cytoplasm. Two camelid single domain antibodies that are folded and soluble when expressed in the cytoplasm have been described (Kirchhofer Al., et al, 2010; Saerens et al., 2005).
Another strategy for producing intracellular antibodies in the bacterial cytosol is the use of E. coli mutants that have mutations that change the redox state of proteins in the cytoplasm from reducing to oxidizing. This produces scFvs that are folded and partially and/or fully oxidized in the E. coli cytoplasm (He et al, 1995; Jurado P., et al., 2002).
Two groups that used the yeast-two-hybrid (Y2H) system as an in vivo screen for scFv binding to antigen from scFv libraries compiled sequences for their soluble clones. The first group (Tse et al., 2002) found that the VH3 Glade was paired with clades VLκ 1 and 4. By aligning multiple soluble scFvs they compiled a consensus for soluble VL and VH genes that almost exactly matched the family consensus compiled for the Morphosys HuCAL™ library for families VH3 and VLκ1 (Knappik et al., 2000). The second group using Y2H reported in WO 03/097697 that their soluble scFvs were either sequences most closely related to members of the VH3, VH1a or VH1b clades combined with sequences most closely related to members of the VLκ1 or VLλ1 or VLλ3 clades. However, their optimal configuration was VLλ3 paired with VH1b. Crucial to note, however, is that none of the sequences reported were exact matches to the translation of the germline sequence of the nearest homologous immunoglobulin gene, with multiple mutations throughout the sequence. This was presumably due to the use by both groups of pre-screened phage libraries to enrich for antigen binding clones before the limiting step of yeast transformation. However, this implies that one, or more, of the mutations in each gene may be conferring a stabilizing effect on scFv folding in the cytoplasm.
To date, there have been no published reports of an intracellular antibody that has an exact identity to the human germline amino acid sequence of the corresponding VL and VH genes. Such an antibody would be an advantageous scaffold for building diversification because it would allow a high yield from cytoplasmic expression, would provide higher stability in oxidized form, would provide greater structural stability ensuring greater tolerance of loop diversification, and would comprise a completely native sequence resulting in lowered patient rejection from production of a full antibody.
We report here the application of a protein display method previously described in WO 2011/075761 to the screening of a human scFv library and the isolation of soluble scFv genes that have identical framework regions to the human germline sequence. Furthermore, we demonstrate remarkable thermostability and tolerance of CDR3 grafting onto the scFv scaffold.